Fluorescence Quenching by Nucleotides of the Plasma Membrane H+-ATPase from Kluyveromyces lactis
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- 作者：José ; G. Sampedro ; Yadira G. Ruiz-Granados ; Hugo Ná ; jera ; Alfredo Té ; llez-Valencia ; Salvador Uribe
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 8, 2007
- 年：2007
- 卷：46
- 期：18
- 页码：5616 - 5622
- 全文大小：215K
- 年卷期：v.46,no.18(May 8, 2007)
- ISSN：1520-4995
文摘
The yeast plasma membrane H+-ATPase isolation procedure was improved; a highly pureenzyme (90-95%) was obtained after centrifugation on a trehalose concentration gradient. H+-ATPasekinetics was slightly cooperative: Hill number = 1.5, S0.5 = 800 
M ATP, and turnover number = 36s-1. In contrast to those of other P-type ATPases, H+-ATPase fluorescence was highly sensitive to nucleotidebinding; the fluorescence decreased 60% in the presence of both 5 mM ADP and AMP-PNP. Fluorescencetitration with nucleotides allowed calculation of dissociation constants (Kd) from the binding site; Kdvalues for ATP and ADP were 700 and 800 M, respectively. On the basis of amino acid sequence andhomology model analysis, we propose that binding of the nucleotide to the N-domain is coupled to themovement of a loop 
structure and to the exposure of the Trp505 residue located in the loop. Therecombinant N-domain also displayed a large hyperbolic fluorescence quenching when ATP binds; however,it displayed a higher affinity for ATP (Kd = 100 M). We propose for P-type ATPases that structuralmovements during nucleotide binding could be followed if a Trp residue is properly located in the N-domain.Further, we propose the use of trehalose in enzyme purification protocols to increase the purity and qualityof the isolated protein and to perform structural studies.
M ATP, and turnover number = 36s-1. In contrast to those of other P-type ATPases, H+-ATPase fluorescence was highly sensitive to nucleotidebinding; the fluorescence decreased 60% in the presence of both 5 mM ADP and AMP-PNP. Fluorescencetitration with nucleotides allowed calculation of dissociation constants (Kd) from the binding site; Kdvalues for ATP and ADP were 700 and 800 M, respectively. On the basis of amino acid sequence andhomology model analysis, we propose that binding of the nucleotide to the N-domain is coupled to themovement of a loop structure and to the exposure of the Trp505 residue located in the loop. Therecombinant N-domain also displayed a large hyperbolic fluorescence quenching when ATP binds; however,it displayed a higher affinity for ATP (Kd = 100 M). We propose for P-type ATPases that structuralmovements during nucleotide binding could be followed if a Trp residue is properly located in the N-domain.Further, we propose the use of trehalose in enzyme purification protocols to increase the purity and qualityof the isolated protein and to perform structural studies.